Anders Josefsson

Imaging, Biodistribution, and Dosimetry of Radionuclide-Labeled PD-L1 Antibody in an Immunocompetent Mouse Model of Breast Cancer.

The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radionuclide Indium-111 ((111)In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of (111)In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Coinjection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of (111)In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of (111)In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy.

(2S)-2-(3-(1-Carboxy-5-(4-[211At]astatobenzamido)pentyl)ureido)-pentanedioic acid for PSMA-Targeted α-Particle Radiopharmaceutical Therapy

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211At-6 treatment. The antitumor efficacy of 211At-6 was evaluated in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. Results: 211At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At-6 in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.

Imaging of Programmed Cell Death Ligand 1: Impact of Protein Concentration on Distribution of Anti-PD-L1 SPECT Agents in an Immunocompetent Murine Model of Melanoma

Programmed cell death ligand 1 (PD-L1) is part of an immune checkpoint system that is essential for preventing autoimmunity and cancer. Recent approaches in immunotherapy that target immune checkpoints have shown great promise in a variety of cancers, including metastatic melanoma. The use of targeted molecular imaging would help identify patients who will best respond to anti-PD-L1 treatment while potentially providing key information to limit immune-related adverse effects. Recently, we developed an antibody-based PD-L1–targeted SPECT agent—111In-diethylenetriaminepentaacetic acid (DTPA)-anti-PD-L1—to identify PD-L1–positive tumors in vivo. To best use such PD-L1–targeted imaging agents, it is important, as a first step, to understand how the signal is affected by different parameters. Methods: We evaluated the impact of protein concentration on the distribution of 111In-DTPA-anti-PD-L1 in a murine model of aggressive melanoma. Results: 111In-DTPA-anti-PD-L1 (dissociation constant, 0.6 ± 0.1 nM) demonstrated increased uptake in B16F10 tumors at protein concentrations equaling or exceeding 1 mg/kg at 24 h and 3 mg/kg at 72 h. At 24 h, the PD-L1–rich spleen and lungs demonstrated decreasing uptake with increasing protein concentration. At 72 h, uptake in the thymus was significantly increased at protein concentrations of 3 mg/kg or greater. Both time points demonstrated increased tracer amounts remaining in circulation as the amount of cold antibody was increased. Conclusion: These studies demonstrate that 111In-DTPA-anti-PD-L1 is capable of identifying tumors that overexpresses PD-L1 and monitoring the impact of PD-L1–rich organs on the distribution of anti-PD-L1 antibodies.

Comparative Dosimetry for 68Ga-DOTATATE: Impact of using Updated ICRP phantoms, S values and Tissue Weighting Factors

The data that have been used in almost all calculations of MIRD S value absorbed dose and effective dose are based on stylized anatomic computational phantoms and tissue-weighting factors adopted by the International Commission on Radiological Protection (ICRP) in its publication 60. The more anatomically realistic phantoms that have recently become available are likely to provide more accurate effective doses for diagnostic agents. 68Ga-DOTATATE is a radiolabeled somatostatin analog that binds with high affinity to somatostatin receptors, which are overexpressed in neuroendocrine tumors and can be used for diagnostic PET/CT-based imaging. Several studies have reported effective doses for 68Ga-DOTATATE using the stylized Cristy–Eckerman (CE) phantoms from 1987; here, we present effective dose calculations using both the ICRP 60 and more updated formalisms. Methods: Whole-body PET/CT scans were acquired for 16 patients after 68Ga-DOTATATE administration. Contours were drawn on the CT images for spleen, liver, kidneys, adrenal glands, brain, heart, lungs, thyroid gland, salivary glands, testes, red marrow (L1–L5), muscle (right thigh), and whole body. Dosimetric calculations were based on the CE phantoms and the more recent ICRP 110 reference-voxel phantoms. Tissue-weighting factors from ICRP 60 and ICRP 103 were used in effective dose calculations for the CE phantoms and ICRP 110 phantoms, respectively. Results: The highest absorbed dose coefficients (absorbed dose per unit activity) were, in descending order, in the spleen, pituitary gland, kidneys, adrenal glands, and liver. For ICRP 110 phantoms with tissue-weighting factors from ICRP 103, the effective dose coefficient was 0.023 ± 0.003 mSv/MBq, which was significantly lower than the 0.027 ± 0.005 mSv/MBq calculated for CE phantoms with tissue-weighting factors from ICRP 60. One of the largest differences in estimated absorbed dose coefficients was for the urinary bladder wall, at 0.040 ± 0.011 mGy/MBq for ICRP 110 phantoms compared with 0.090 ± 0.032 mGy/MBq for CE phantoms. Conclusion: This study showed that the effective dose coefficient was slightly overestimated for CE phantoms, compared with ICRP 110 phantoms using the latest tissue-weighting factors from ICRP 103. The more detailed handling of electron transport in the latest phantom calculations gives significant differences in estimates of the absorbed dose to stem cells in the walled organs of the alimentary tract.

Re-evaluation of pediatric 18F-FDG dosimetry: Cristy–Eckerman versus UF/NCI hybrid computational phantoms

Because of the concerns associated with radiation exposure at a young age, there is an increased interest in pediatric absorbed dose estimates for imaging agents. Almost all reported pediatric absorbed dose estimates, however, have been determined using adult pharmacokinetic data with radionuclide S values that take into account the anatomical differences between adults and children based upon the older Cristy-Eckerman (C-E) stylized phantoms. In this work, we use pediatric model-derived pharmacokinetics to compare absorbed dose and effective dose estimates for 18F-FDG in pediatric patients using S values generated from two different geometries of computational phantoms. Time-integrated activity coefficients of 18F-FDG in brain, lungs, heart wall, kidneys and liver, retrospectively, calculated from 35 pediatric patients at the Bostons Children Hospital were used. The absorbed dose calculation was performed in accordance with the Medical Internal Radiation Dose method using S values generated from the University of Florida/National Cancer Institute (UF/NCI) hybrid phantoms, as well as those from C-E stylized computational phantoms. The effective dose was computed using tissue-weighting factors from ICRP Publication 60 and ICRP Publication 103 for the C-E and UF/NCI, respectively. Substantial differences in the absorbed dose estimates between UF/NCI hybrid pediatric phantoms and the C-E stylized phantoms were found for the lungs, ovaries, red bone marrow and urinary bladder wall. Large discrepancies in the calculated dose values were observed in the bone marrow; ranging between  -26% to  +199%. The effective doses computed by the UF/NCI hybrid phantom S values were slightly different than those seen using the C-E stylized phantoms with percent differences of  -0.7%, 2.9% and  2.5% for a newborn, 1 year old and 5 year old, respectively. Differences in anatomical modeling features among computational phantoms used to perform Monte Carlo-based photon and electron transport simulations for 18F, and very likely for other radionuclides, impact internal organ dosimetry computations for pediatric nuclear medicine studies.

Dosimetry and Radiobiology of Alpha-Particle Emitting Radionuclides

BACKGROUND Radiopharmaceutical therapy is a cancer treatment modality by which radiation is delivered directly to targeted tumor cells or to their microenvironment. This makes it possible to deliver highly potent alpha-particle radiation. The short-range and highly potent nature of alpha-particles require a dosimetry methodology that considers microscale distributions of the alpha-emitting agent. The high energy deposition density along an alpha-particle track causes a spectrum of DNA lesions. The majority of these are irreparable DNA double-stranded breaks. Accordingly the biologic effects of alpha- particles are largely impervious to the adaptive and resistance mechanism that renders other therapeutics ineffectual. OBJECTIVES In this review, the radiobiology and dosimetry of alpha-particle emitting radionuclides as related to their use in radiopharmaceutical therapy, are presented. CONCLUSION Alpha-particle emitter radiopharmaceutical therapy is distinguished from other treatment modalities. Its safe clinical use requires an understanding of its unique dosimetry and radiobiology.

Human HER2 overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer

Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-particle emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from primary tumors and lung metastases from HuHER2 transgenic mice. We extracted primary mammary gland tumors, isolated the epithelial breast cancer cells, and established seven different cell lines. We also established 2 different cell lines from spontaneous lung metastases and cell lines from a serial transplantation of tumor tissues in HuHER2 transgenic mice. HuHER2 protein was overexpressed in all of the established cell lines. The mRNA level of ER (estrogen receptor) and PR (progesterone receptor) was relatively low in the cell lines compared to normal mammary gland (MG). As EMT markers, the expression of E-Cadherin in the cell lines was downregulated while the expression of TWIST1 and Vimentin were upregulated, relative to MG. Furthermore, trastuzumab directly inhibited cellular viability. Biodistribution studies with 111In-DTPA-trastuzumab in HuHER2 cell tumor xenografts demonstrated specific targeting with a clinically relevant antibody. Collectively, these cell lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies.Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-particle emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from primary tumors and lung metastases from HuHER2 transgenic mice. We extracted primary mammary gland tumors, isolated the epithelial breast cancer cells, and established seven different cell lines. We also established 2 different cell lines from spontaneous lung metastases and cell lines from a serial transplantation of tumor tissues in HuHER2 transgenic mice. HuHER2 protein was overexpressed in all of the established cell lines. The mRNA level of ER (estrogen receptor) and PR (progesterone receptor) was relatively low in the cell lines compared to normal mammary gland (MG). As EMT markers, the expression of E-Cadherin in the cell lines was downregulated while the expression of TWIST1 and Vimentin were upregulated, relative to MG. Furthermore, trastuzumab directly inhibited cellular viability. Biodistribution studies with 111In-DTPA-trastuzumab in HuHER2 cell tumor xenografts demonstrated specific targeting with a clinically relevant antibody. Collectively, these cell lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies.

Abstract 3052: Combining α-particle radiopharmaceutical therapy using Actinium-225 and immunotherapy with anti-PD-L1 antibodies in a murine immunocompetent metastatic breast cancer model

The programmed cell death ligand 1 (PD-L1) plays an essential role in suppressing immune recognition of cancer. PD-L1 is expressed on a variety of cells including tumor cells, tumor associated macrophages (TAMs) and other cells within the microenvironment of the tumor. When PD-L1 binds to the programmed death 1 (PD-1) receptor it inhibits CD8+ T-cell effector function. By upregulating the expression levels of PD-L1, tumor cells and TAMs are capable of avoiding T-cell immune recognition. Immunotherapy using anti-PD-L1 antibody (Ab) has shown promising anti-tumor effect against a number of cancers including breast cancer, and is currently used in several clinical trials. Furthermore, studies have shown that anti-PD-L1 Ab targeted immunotherapy synergizes with radiation therapy. The aim of this study was to investigate a possible gain in therapeutic efficacy when combining targeted α-particle radiopharmaceutical therapy using 225Ac with anti-PD-L1 Ab immunotherapy in a murine immunocompetent metastatic breast cancer model. 6-8 week old healthy female neu-N mice were injected in the left cardiac ventricle (LCV) with 50,000 NT2.5 (endogenously derived) tumor cells to create highly aggressive widespread breast cancer metastases. Groups (n = 8) of mice were injected intravenously (i.v.) in the tail vein 72 h after the LCV injection with either 1) a single dose of 300 or 400 nCi 225Ac-DOTA-anti-PD-L1 Ab (0.15 mg/kg), 2) a single dose of 100 times (100x) anti-PD-L1 Ab (15.9 mg/kg) or 3) a 400 nCi single dose 225Ac-DOTA-anti-PD-L1 Ab (0.15 mg/kg) in combination with a single dose of 100x anti-PD-L1 Ab (16.1 mg/kg). The mice in the control group were injected i.v. in the tail vein with 100 μl of saline. The 225Ac-DOTA-anti-PD-L1 conjugate was radiolabeled having a specific activity of 0.137 μCi/μg with a radiochemical purity >95%. The group receiving the single dose of 100x anti-PD-L1 Ab had the highest median survival of 44 days (p = 0.0007) followed by the 400 nCi 225Ac-DOTA-anti-PD-L1 Ab group with 39.5 days (p = 0.0413) compared with the control group 31.5 days. The survival for other treatment groups were not significant compared with the control group. Furthermore, the survival from the single dose of 100x anti-PD-L1 Ab treatment was significantly higher than the single dose treatment of 400 nCi 225Ac-DOTA-anti-PD-L1 Ab (p = 0.0308). The highest survival rate was the mice treated with 100x anti-PD-L1 Ab. The combination treatment using 400 nCi 225Ac-DOTA-anti-PD-L1 Ab and 100x anti-PD-L1 Ab showed a significant lower survival compared to each treatment by itself. However, the combination treatment was only performed with one dose and the injection was at the same time, different concentrations and time spaced injections could possibly favor the combined treatment method over the single modality treatments. Citation Format: Anders Josefsson, Jessie R. Nedrow, Sunju Park, Sagar Ranka, George Sgouros. Combining α-particle radiopharmaceutical therapy using Actinium-225 and immunotherapy with anti-PD-L1 antibodies in a murine immunocompetent metastatic breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3052.

Abstract LB-183: The impact of protein concentration on the distribution of 111In-antibody conjugate for imaging of programmed death-ligand 1 (PD-L1)

Background: Programmed cell Death Ligand 1 (PD-L1) is part of an immune checkpoint system that is essential for preventing autoimmunity. Tumor cells have developed the ability to co-opt these immune checkpoints to suppress anti-tumor immunity. Recent approaches in immunotherapy targeting immune checkpoints have shown great promise in a variety of cancers. PD-L1 overexpression is associated with a poorer prognosis in a variety of cancers, but typically have a stronger response to anti-PD-L1 therapy. PD-L1 is a dynamic biomarker and the ability to image its dynamic nature could provide key information in identifying patients who will best respond to anti-PD-L1 treatment with limited adverse effects. Here we evaluated an 111In-antibody conjugate targeted to PD-L1 for SPECT imaging in an aggressive mouse melanoma model. In addition, we investigated the relationship between the spleen, which we have found to be a sink for anti-PD-L1 antibodies, and specific activity (activity/protein concentration) of the 111In-antibody conjugate. Methods: An anti-PD-L1 antibody was conjugated to DTPA for 111In-labeling. The biodistribution of the resulting conjugate, 111In-DTPA-anti-PD-L1, was performed over a range of specific activities in B16F10 tumor bearing mice. SPECT imaging was also performed with the 111In tracer. Results: 111In-DTPA-anti-PD-L1 (Kd = 0.6±0.1 nM) having a specific activity (SA) of 0.57 Ci/μmol demonstrated uptake in the B16F10 tumor (6.1±2.8%ID/g) at 24 hours, as well as high uptake in the spleen (32.8±5.1%ID/g). Decreasing the SA to 0.01 Ci/μmol blocked PD-L1-postive cells in the spleen (13.4±4.8%ID/g) forcing more of the tracer to remain in circulation, allowing for increase accumulation in the B16F10 tumor (16.8±6.2%ID/g) at 24 hours. The lower SA also demonstrated a reduction in tumor to blood ratios compared to the higher SA (2.2 vs. 4.1), but an increase was shown in the tumor to muscle ratio (27 vs. 4.6). Conclusion: 111In-DTPA-anti-PD-L1 is capable of identifying tumors that overexpresses PD-L1, and has the potential to help select patients that will best respond to anti-PD-L1 therapy. In addition, the further understanding of the spleen/tumor relationship could be utilized to tailor individual doses for anti-PD-L1 therapy and minimize adverse effects. Citation Format: Jessie Nedrow, Anders Josefsson, Sunju Park, Sagar Ranka, George Sgouros. The impact of protein concentration on the distribution of 111In-antibody conjugate for imaging of programmed death-ligand 1 (PD-L1). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-183.